Fluorescence recovery under decaying photobleaching irradiation: concept and experiment.

A novel modification of photobleaching method for measurement of lateral diffusion is developed. In this approach fluorescence recovery kinetics is measured under decaying photobleaching irradiation, termed as fluorescence recovery under decaying photobleaching (FRDP). The time evolution of fluorescence intensity normalized to input irradiation starts from the photobleaching kinetics and transforms into the kinetics of fluorescence recovery at a later stage resulting in appearance of minimum. The analytical solution for the kinetics of fluorescence for Gaussian lineshape of laser beam and hyperbolic decay of irradiation in the first order approximation on bleaching rate was obtained. The accuracy of the analytical function was evaluated with exact numerical solution computed with finite differentiates method. The FRDP method was successfully applied to fluorescein solution in the glycerol/water mixture (80%) under various experimental settings using home-made experimental set-up. The FRDP approach demonstrated 25-30 fold enhancement in signal intensity over classical fluorescence recovery after photobleaching (FRAP) method at 3-5 fold increase in total irradiation. Among other advantages of the FRDP is the opportunity to perform measurements on varying time scales under constant size of the bleaching spot, including "safe" long time measurements. The potential extra advantage of FRDP method for analysis of complex diffusion in the biological system is discussed.